Method of identifying compounds that induce cell death by necrosis

ABSTRACT

The present invention provides methods of identifying and/or detecting anti-cancer agents. The present invention provides methods of identifying and/or detecting compounds that can activate PARP and/or induce necrosis. The present invention also provides for methods of treating cancer in an individual. The present invention also provides kits for identifying and/or detecting anti-cancer agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/529,642, filed Dec. 15, 2003, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Cell death plays an important role in development, tissue homeostasis,and degenerative diseases. Two major forms of cell death have beendescribed: apoptosis and necrosis. Apoptosis, also called programmedcell death, is an energy-driven process by which a cell activelydestroys itself in response to extracellular signals or developmentalcues, whereas necrosis has been considered a passive process in which acell dies as a result of bioenergetic catastrophe. Apoptosis ischaracterized by the ordered cellular degradation of proteins andorganelles, maintenance of plasma membrane integrity, andnon-inflammatory phagocytosis of the dying cell (Adams 2003; Wang 2001).During necrosis, cells swell rapidly and lose the integrity of theirplasma membrane, releasing cellular contents into the extracellularenvironment, and triggering an acute inflammatory response. Necrosis hastraditionally been considered an unregulated form of cell death, and hasbeen well characterized in a wide range of pathologic states includingischemia, trauma, and infection (Majno and Joris 1995; Kanduc et al.2002).

A great deal of recent attention has focused on the role of apoptosis innormal development and various disease processes. Most if not all cancercells have defects in the normal control of apoptosis. The firstcharacterized example of this is the 14:18 chromosomal translocationfound in patients with follicular lymphoma that juxtaposes theimmunoglobulin enhancer with the anti-apoptotic gene bcl-2 (Tsujimoto etal. 1984). Enhanced expression of Bcl-2 provides resistance to apoptosisby suppressing the activation of the proapoptotic Bcl-2 related proteinsBax and Bak. Bax and Bak are essential in apoptosis initiated from bothmitochondria and the endoplasmic reticulum (ER). Cells lacking both Baxand Bak are resistant to apoptosis induced by developmental cues, signaltransduction through death receptors, growth factor withdrawal, and ERstress (Lindsten et al. 2000; Wei et al. 2001; Zong et al. 2001, Chenget al. 2001; Degenhardt et al. 2002b; Scorrano et al. 2003; Zong et al.2003).

Despite the role of Bcl-2 as an anti-apoptotic protein, follicularlymphoma cells are sensitive to treatment with DNA alkylating drugs invivo (Lister 1991).

Although there have been advancements in the treatment of cancer ahallmark of many cancers is that the treatments fail to work after aperiod of time as the cancer cells become resistant to the treatment.Much of this resistance is due to inhibition of the apoptosis pathwaydue to genetic mutations in the cell. However, cells can still die byother processes including necrosis. Thus, there is a need to identifycompounds that can act as anti-cancer agents. There is also a need toidentify anti-cancer agents that are able to kill cancer cells that donot die by apoptosis.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides methods of detectingan anti-cancer agent comprising performing a test assay comprisingcontacting an immortalized cell with a test compound and measuring PARPactivity.

In some embodiments, the present invention provides kits for identifyinga PARP activator comprising

In some embodiments, the present invention provides methods of treatingcancer in an individual comprising identifying an anti-cancer agent andadministering the agent to the individual.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1, Panels A-D show that cells deficient in p53 or Bax/Bak aresusceptible to DNA alkylating agents. MEFs from wild-type, p53^(−/−),and bax^(−/−)bak^(−/−) mice were treated with staurosporine (Panel A),etoposide (Panel B), nitrogen mustard (Panel C), and MNNG (Panel D) asdescribed in Materials and Methods. Drug concentrations were indicatedin the individual panels. Cell survival was determined for triplicatesamples by PI exclusion at 20 h following treatment. Data is presentedas mean+/−S.D., and is representative of three independent experiments.

FIG. 2, Panels A-C show that alkylating DNA damage results in PARPactivation and bioenergetic collapse. Panel A: MNNG activates PARP inboth wild-type and bax^(−/−)bak^(−/−) cells. Wild-type andbax^(−/−)bak^(−/−) MEFs were treated with MNNG (0.5 mM) for theindicated periods of time. Cells were lysed and immunoblotting wasperformed using an antibody against poly(ADP-ribose) (PAR). Asteriskmarks a non-specific band. Panels B and C: Depletion of NAD and ATP inresponse to PARP activation. Wild-type and bax^(−/−)bak^(−/−) cells weretreated with MNNG at indicated concentrations for 30 min, alone ortogether with PARP inhibitor DPQ. The cellular NAD (Panel B) and ATP(Panel C) levels were determined. Concentrations of NAD and ATP werenormalized with that of untreated cells.

FIG. 3, Panels A-G, show that PARP inhibition results in resistance toMNNG-induced cell death. Panel A: PARP-1 shRNA in bax^(−/−)bak^(−/−)cells. Stable clones were selected from bax^(−/−)bak^(−/−) MEFstransfected with vector or PARP-1 hairpins. Cell lysates were made fromvector cell line or PARP-1 hairpin cell lines. Immunoblotting wasperformed using an anti-PARP-1 antibody, and an anti-Tom20 antibody as acontrol for equal loading. Note that PARP-1 expression was suppressedsignificantly in Clone HP17, moderately in HP11, and not effected inHP23. Panel B: PARP activity was determined using triplicate samples byimmunoblotting using an anti-PAR antibody. Panel C: ATP levels weremeasured after 30 min treatment with MNNG at indicated concentrations.Panel D: Cell survival was determined by PI exclusion 20 h after MNNGtreatment. Panels E and Panel F: Wild-type, bax^(−/−)bak^(−/−),parp-1^(−/−), and Clone HP17 (bax^(−/−)bak^(−/−), shPARP-1) MEFs weretreated with MNNG (0.5 mM) alone or together with PARP inhibitor DHIQ,or in the presence of staurosporine (STS). 24 h later cells werephotographed under a phase-contrast filter (Panel E), and cell survivalwas determined by PI exclusion (Panel F). Panel G: Wild-type,bax^(−/−)bak^(−/−), parp-1^(−/−), and Clone HP17 MEFs were treated withMNNG (0.25 mM). Cell survival was measured over time by PI exclusion.

FIG. 4, Panels A-C show PARP-mediated cell death is necrotic. Panel A:Wild-type and bax^(−/−)bak^(−/−) cells were treated with 0.5 mM MNNG, or2 μM staurosporine (STS). Transmission electron microscopy was performed9 h later. Panel B: Transformed wild-type baby mouse kidney (BMK) cellswere treated with MNNG alone or together with DPQ. Immunofluorescencewas performed 6 h later using an antibody against cytochrome c. Cellswere counter-stained with DAPI to show the nuclear morphology. Panel C:Wild-type and bax^(−/−)bak^(−/−) MEFs were treated with indicatedagents. Cells were lysed after 8 h. 20 μg of protein was separated on a4-12% gradient NuPAGE gel. PARP and Lamin B1 were detected usingrespective antibodies.

FIG. 5, Panels A-C show PARP-mediated cell death is pro-inflammatory.Panel A: HMGB1 translocates from nucleus into cytosol upon MNNGtreatment. bax^(−/−)bak^(−/−) MEFs were treated with 0.5 mM MNNG. 6 hlater immunofluorescence was performed using an antibody against HMGB1.Nuclei were visualized by DAPI staining. Panel B: HMGB1 is released intoextracellular environment during MNNG-induced necrosis. Wild-type andbax^(−/−)bak^(−/−) MEFs were treated with 0.5 mM MNNG alone or togetherwith DPQ. 16 h later culture media were collected, and cells lysed inRIPA buffer. HMGB1 was detected by immunoblotting in both cell lysatesand culture media. Panel C: Inflammatory response triggered by necrosis.Wild-type, bax^(−/−)bak^(−/−), or parp-1^(−/−) MEFs were treated withMNNG (0.5 mM) for 15 min or STS (2 μM) for 2 h. Drugs were washed awayand cells refed with fresh media. 20 h later, cell culture media werecollected and added to 1×10⁵ macrophages. Concentration of TNFα wasmeasured 24 h later.

FIG. 6, Panels A-E show vegetative cells are more resistant toPARP-mediated necrosis. Panel A: IL-3-dependent hematopoieticbax^(−/−)bak^(−/−) cells were cultured in media with or without IL-3 for2 d. Cells were treated with MNNG for 15 min at indicated concentrationsand cell survival was measured by PI exclusion 24 h later. Panel B:Wild-type and bax^(−/−)bak^(−/−) MEFs were plated at 2×10⁴/well(subconfluent) or 2×10⁵/well (confluent) in 12-well plates. Cells werecultured for 36 h. Confluent cells were then cultured in the absence ofserum for 12 h and subconfluent cells cultured in the presence of serum.Cells were treated with 0.5 mM MNNG for 15 min. Cell survival wasdetermined 24 h later by PI exclusion. Panels C and D: IL-3-dependentbax^(−/−)bak^(−/−) cells were cultured in the presence or absence ofIL-3 for 2 d. Cells were then treated with MNNG for 15 min. PARPactivity was determined by immunoblotting of PAR, and NAD and ATP levelsmeasured and shown as the percentage of the levels in untreated cells.(Panel C). Cellular NAD pool was decreased in both proliferating andvegetative cells, while cellular ATP pool was preserved in vegetativecells but not in proliferating cells (Panel D). Panel E: PARP activationpreferentially depletes cytosolic NAD. IL-3-dependent cells were treatedwith 0.5 mM MNNG for 15 min. Cells were fractionated and the NAD levelswere measured in total cell lysates, as well as in the cytosolic andmitochondrial fractions. Immunoblotting of tubulin and COX IV wereperformed to assure the purity of the fractionation.

FIG. 7, Panels A-D show susceptibility to PARP-mediated necrosis iscontrolled by cellular metabolic status. Panel A: Cellular glycolysisrate in the presence or absence of IL-3. IL-3-dependentbax^(−/−)bak^(−/−) cells were cultured in the presence or absence ofIL-3 for 2 d. One million cells were harvested and their glycolysis ratedetermined. Panel B: Effect of inhibition of oxidative phosphorylationon ATP levels in cells cultured with or without IL-3. Cells cultured inmedia with or without IL-3 were treated with oligomycin (5 μg/ml) for 30min, and ATP levels determined. Panel C: IL-3-dependentbax^(−/−)bak^(−/−) cells cultured in the presence or absence of IL-3 for2 d. An additional population was cultured in the presence of IL-3 incomplete medium made without glucose (−glucose), while a fourthpopulation was cultured in the presence of IL-3 and supplemented with 10mM methyl-pyruvate (+pyruvate) immediate prior to MNNG treatment. Cellswere then treated with MNNG and cell survival determined by PI exclusionas described in Materials and Methods. Data presented is representativeof 3 independent experiments. Panel D: Methyl-pyruvate (10 mM) was addedto the IL-3-dependent wild-type cells immediate prior to MNNG treatment.Cells were treated with MNNG and cell survival determined by PIexclusion.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Programmed cell death has been defined as cell suicide in response todevelopmental cues or intrinsic cell stress. The major form ofprogrammed cell death described to date is apoptosis. Apoptotic celldeath is ATP-dependent, results in organized proteolytic degradation ofthe intracellular contents, and phagocytosis of the dying cell withoutinducing an inflammatory response. Described herein is a form of celldeath in which the cell actively initiates its own necrosis in responseto DNA damage (e.g. by an alkylation agent). This form of cell death isindependent of the major apoptotic effectors (e.g. p53, Bax, Bak, andcaspases).

In response to DNA damage, cells activate PARP, an enzyme that catalyzespoly(ADP-ribosyl)ation of a variety of proteins (D'Amours et al. 1999).This activity of PARP can increase the accessibility of DNA to DNArepair enzymes and transcription factors. There has been controversyconcerning the role of PARP in the regulation of cell survival/death inresponse to DNA damage. Some work has implicated PARP in the regulationof DNA repair and cell survival (Wang et al. 1997), while others haveimplicated PARP in initiating cell death by either apoptosis (Yu et al.2002) or necrosis (Ha and Snyder 1999). It has been observed that theactivation of PARP runs a metabolic test on the damaged cell. In cellsthat maintain their ATP production exclusively by catabolizing glucose,PARP activation results in a rapid decline in cellular ATP and necroticdeath. In contrast, in cells that can maintain non-glucose-dependentoxidative phosphorylation, PARP activation does not compromise cellularbioenergetics and such cells are resistant to DNA damage-induced death.Accordingly, activation of PARP can be used to kill cell, particularlycancer cells, which rely on catabolizing glucose to maintain their ATPproduction.

The cellular test run by PARP provides a potential explanation for theability of DNA alkylating drugs to act as effective chemotherapeuticagents. In general it is believed that most chemotherapeutic drugsinduce tumor cells to die by apoptosis. However, a central feature ofperhaps all cancers is the development of apoptotic resistance. The mostcommon genetic abnormality in human tumors is mutation of p53. Loss ofp53 function is associated with pronounced apoptotic resistance (Vousdenand Lu 2002; Gudkov and Komarova 2003).

Nevertheless, alkylating agents remain the single most effective andbroadly active chemotherapeutic agents (Chabruer and Longo 2001). It isthought that the efficacy of alkylating drugs results from the selectiveability to kill proliferating cells. Proliferating cells have been shownto have an increased propensity to apoptosis under some circumstances(Evan and Vousden 2001). However, the discovery that neither Bax/Bakdeficiency nor p53 deficiency is required to affect cell death inresponse to DNA damage suggested that there might be alternativeexplanations for the ability of alkylating agents to selectively killtumor cells such as by PARP-mediated necrosis.

Tumor cells display an abnormal propensity for growth and are in netprotein and lipid synthesis. As a result, they maintain ATP productionalmost exclusively through catabolizing glucose through a mixture ofglycolysis and oxidative phosphorylation termed aerobic glycolysis (Holt1983; Baggetto 1992). In contrast, cells that are not actively growingor replicating are capable of catabolizing a variety of metabolicsubstrates to maintain ATP production (Eaton et al. 1996). Inhibition ofmitochondrial respiratory chain has been shown to induce death ofvegetative cells (Hartley et al. 1994; Woodgate et al. 1999; Ohgoh etal. 2000). In contrast, mitochondrial inhibitors do not affect theviability of cells with a high level glycolytic rate because they arecapable of maintaining ATP levels without mitochondrial respiration(Shchepina et al. 2002). By differentially affecting ATP generation fromglycolysis or mitochondrial respiration, activation of PARP canselectively kill cells that are preferentially maintaining theirbioenergetics through glycolysis. The proliferating cell's dependency onaerobic glycolysis that determines their sensitivity to PARP-inducedcell death. This necrotic form of programmed cell death may be adaptiveas it both eliminates the possibility of cell survival and induces animmune response to the dying cell. In response to DNA damage, there isan active redistribution of the macrophage activator HMGB1 from thenucleus to the cytosol. HMGB1 is a small acidic protein localized tochromatin primarily by electrostatic forces (Butler et al. 1985). Ashistones and other chromatin proteins are modified bypoly(ADP-ribosyl)ation, the resulting increase in negative charge maydisplace HMGB1, thus releasing it from the chromatin. This in turn willincrease HMGB1 release into the extracellular environment if the cellloses plasma membrane integrity during necrotic death. In contrast,HMGB1 has been shown to have increased affinity for the DNA fragmentscreated during apoptosis (Scaffidi et al. 2002). This increasedsequestration of HMGB1 suppresses its ability to be released from thecell during apoptosis. PARP-dependent release of proinflammatorymediators such as HMGB1 distinguishes programmed necrosis fromapoptosis.

The ability of alkylating agent-induced necrosis to induce aninflammatory response to the dying tumor cells may contribute to theefficacy of the drugs as chemotherapeutic agents. Consistent with animportant role for PARP-mediated necrosis in inflammatory responses,parp-1^(−/−) mice are resistant to ischemia-reperfusion (Eliasson et al.1997), endotoxic shock (Szabo et al. 1996), and streptozotocin-induceddiabetes (Burkart et al. 1999; Masutani et al. 1999, Pieper et al.1999a).

Thus, necrosis can be a regulated cell fate independent of apoptosis.Necrotic cell death following DNA damage occurs selectively in cellscommitted to growth. This prevents the survival of cells at risk ofaccumulating mutations as a result of DNA replication prior to DNArepair. In addition, this form of programmed necrosis induces aninflammatory response that provides additional protection against theaccumulation of damaged or aberrant cells, and initiates the repair ofthe damaged tissue.

Accordingly, the present invention arises out of the discovery that theactivation of poly(ADP-ribose) polymerase (PARP) in a tumor cell, animmortalized cell, or a cell committed to growth by the catabolism ofglucose leads to cell death that is independent of the apoptoticmachinery. This discovery can be used to identify test compounds thatcan activate PARP and thus, can be used as anti-cancer compounds.

The present invention provides methods of detecting or identifying ananti-cancer agent. In some embodiments, the methods comprise contactinga cell with a test compound and measuring PARP activation. The cell thatis contacted with the test compound can be any cell including, but notlimited to, a tumor cell, an immortalized cell, an undifferentiatedcell, and the like.

As used herein, the term “tumor cell” refers to a cell that has beenderived from a tumor. The tumor cell can be from a primary tumor or itcan be from a tumor that has metastasized. The tumor cell can also befrom a tumor cell line. Tumor cell lines are widely available and can beobtained from many companies including, but not limited to, ATCC(American Type Culture Collection, Rockville, Md.).

As used herein, the term “immortalized cell” refers to a cell that doesnot under normal growth conditions undergo quiescence. An “immortalizedcell” can in some embodiments, be a tumor cell. “Immortalized cells” canalso be normal cells that have been transformed to become immortal.Examples of cells that can be immortalized include, but not limited to,embryonic fibroblasts, which include mouse embryonic fibroblasts (MEFs).Mouse embryonic fibroblasts undergo what is termed “crisis” that allowsthem to become immortalized.

As used herein, the term “undifferentiated cell” refers to a cell thatcan become differentiated or has the ability to become different typesof cells depending on its environment and/or signals that the cellreceives.

The test compound can be contacted with a cell by an means that isavailable that puts the compound in contact with the cell. In someembodiments, the test compound is injected into the cell. If the cell isin an in vitro environment (e.g. cell culture) the test compound can beadded to the media that the cell is growing in. The test compound canalso be tested in vivo by administering the test compound to an animal.The test compound can be administered by any means available including,but not limited to, injection, orally, and the like.

In some embodiments, the methods of the invention comprises contacting atest compound with the cell population under particular conditions andmeasuring PARP activation in the cells, as an indication of the effectof the test compound. In some embodiments, it is determined if the cellshave undergone necrosis and is used as an indication of the effect ofthe test compound. In some embodiments, the effect of the test compoundis compared what occurs in the absence of any test compound.

In some embodiments the methods comprise contacting more than one testcompounds, in parallel. In some embodiments, the methods comprisescontacting 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100,1000, at least 2, at least 5, at least 10, at least 50, at least 100, orat least 1000 test compounds in parallel. In some embodiments, thepresent invention is used High Throughput Screening of compounds andcomplete combinatorial libraries can be assayed, e.g., up to thousandsof compounds. Methods of how to perform high throughput screenings arewell known in the art. The methods can also be automated such that arobot can perform the experiments. The present invention can be adaptedfor the screening of large numbers of compounds, such as combinatoriallibraries of compounds. Indeed, compositions and methods allowingefficient and simple screening of several compounds in short periods oftime are provided. The instant methods can be partially or completelyautomated, thereby allowing efficient and simultaneous screening oflarge sets of compounds.

In some embodiments, the method of the present invention comprises thestep of contacting a cell in the presence of a test compound. The cellscan then be observed to determine if the test compound activates PARP orcauses necrosis. A control may be provided in which the cell is notcontacted with a test compound. A further control may be provided inwhich test compound is contacted with cells that either do not expressfunctional PARP or in which PARP is inactivated. If the cells contactedwith the test compound activate PARP, cause necrosis, or both thenanti-cancer activity is indicated for the test compound.

Positive and negative controls may be performed in which known amountsof test compound and no compound, respectively, are added to the assay.One skilled in the art would have the necessary knowledge to perform theappropriate controls.

The test compound can be any product in isolated form or in mixture withany other material (e.g., any other product(s)). The compound may bedefined in terms of structure and/or composition, or it may beundefined. For instance, the compound may be an isolated andstructurally-defined product, an isolated product of unknown structure,a mixture of several known and characterized products or an undefinedcomposition comprising one or several products. Examples of suchundefined compositions include for instance tissue samples, biologicalfluids, cell supernatants, vegetal preparations, etc. The test compoundmay be any organic or inorganic product, including a polypeptide (or aprotein or peptide), a nucleic acid, a lipid, a polysaccharide, achemical product, or any mixture or derivatives thereof. The compoundsmay be of natural origin or synthetic origin, including libraries ofcompounds.

In some embodiments, the activity of the test compound(s) is unknown,and the method of this invention is used to identify compoundsexhibiting the selected property (e.g., PARP activation or necrosisinducing). However, in some embodiments instances where the activity (ortype of activity) of the test compound(s) is known or expected, themethod can be used to further characterize the activity (in terms ofspecificity, efficacy, etc.) and/or to optimize the activity, byassaying derivatives of the test compounds.

The amount (or concentration) of test compound can be adjusted by theuser, depending on the type of compound (its toxicity, cell penetrationcapacity, etc.), the number of cells, the length of incubation period,etc. In some embodiments, the compound can be contacted in the presenceof an agent that facilitates penetration or contact with the cells. Thetest compound is provided, in some embodiments, in solution. Serialdilutions of test compounds may be used in a series of assays. In someembodiments, test compound(s) may be added at concentrations from 0.01μM to 1M. In some embodiments, a range of final concentrations of a testcompound is from 10 μM to 100 μM. One such test compound that iseffective to activate PARP activity is a DNA damaging agent thatalkylates DNA.

In some embodiments, the method comprises measuring PARP activation inthe presence of the test compound. If the test compound is found to be aPARP activator it is indicative that the test compound is an anti-canceragent. PARP activation can be measured by any means that demonstratesthat the activity of the enzyme PARP has been modulated (increased ordecreased) in the presence of the test compound. Examples of how tomeasure PARP activity include measuring an increase of poly(ADP-ribose)polymers (PAR). Other examples of how to measure PARP activity include,but are not limited to, measuring NAD levels and/or ATP levels. In someembodiments, the levels of NAD are depleted in the presence of the testcompound. In some embodiments the levels of ATP are depleted in thepresence of the test compound. Methods of measuring NAD and/or ATPlevels are routine. Methods of measuring the levels of thepoly(ADP-ribose) polymers are routine to one of ordinary skill in theart.

In some embodiments, the test compound activates PARP by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 200%. Insome embodiments, the percent activation of PARP is compared PARPactivity observed in the absence of the test compound.

As described above, the test compound can be contacted with a variety ofcells to determine if it is a PARP activator, necrosis inducing agent,and/or an anti-cancer agent. In some embodiments, the cell that iscontacted with the test compound is unable to undergo apoptosis. In someembodiments the cell is deficient in the expression of the Bax gene, Bakgene, or both.

As used herein, the term “deficient in the expression of” refers to thegene or the product of a gene. The term “deficient in the expression of”can refer to status of the gene in the cell. In some embodiments, thecell is null for the gene in that it has no copies of the gene and is,therefore unable to express the gene. In some embodiments, the status ofthe gene or gene product is that it is mutated such that the gene is notexpressed or that the gene product is not functional or has lessfunction than the wild-type gene. Accordingly, a cell that is deficientin the expression of the Bax gene may have no Bax gene or the Bax genemay be mutated so that the Bax gene product is not functional or hasless function than the wild-type gene.

In some embodiments, the cell that is contacted with the test compoundis null for the Bax gene, Bak gene, or both. A non-limited example of acell that is deficient for the expression of the Bax gene, Bak gene, orboth is a mouse embryonic fibroblast that is deficient in bax and bakgene expression (Zong, et al., Genes & Development, 18:1272-1282(2004)). This cell line (ATCC patent designation number PTA-9386,deposited Jul. 23, 2008) is also described in U.S. Patent Application20030091982, filed May 15, 2003, which is hereby incorporated byreference. However, any cell can be used that is deficient for the Baxgene, Bak gene, or both.

In some embodiments, the cell that is contacted with the test compoundis deficient in p53 gene expression. In some embodiments, a cell that isdeficient in p53 gene expression can have the p53 gene deleted or be“null for p53” or the cell can comprise a mutant of p53 that inactivatesthe function of p53. In some embodiments, the p53 mutant is a dominantnegative mutant or a temperature sensitive mutant. In some embodiments,the p53 mutation is mutation inhibits the binding of p53 to mdm2. Insome embodiments, the p53 mutation inhibits the formation of a p53tetramer.

Methods of creating a cell that is deficient in the expression of aparticular gene or set of genes are known in the art. Examples include,but are not limited to those described in U.S. Patent Application20030091982, siRNA, antisense oligonucleotides, and the like.

In some embodiments, the method comprise determining if a cell hasundergone necrosis. One of skill in the art can determine if a cell hasundergone necrosis by, for example, analyzing the physicalcharacteristics of the cell. Methods of determining if a cell hasundergone necrosis are known to those of skill in the art. Examples ofhow necrosis is determined include, but are not limited to, measuringorganelle swelling, intracellular vacuole formation, plasma membranedisintegration, and nuclear degradation without condensation.

In some embodiments, the methods further comprise performing a controlassay. In some embodiments, the control assay comprising contacting acell with a negative or positive control and measuring, including, butnot limited to, PARP activation, necrosis, and the like. In someembodiments, the control compound is compared to the test compound. Insome embodiments, the control compound is a negative control (e.g. acompound that does not activate PARP, induce necrosis, and/or is not ananti-cancer agent). A negative control can also be the absence of a testcompound or the vehicle (e.g. solvent) that the test compound iscontacted with the cell. In some embodiments, the control compound is apositive control (e.g. a compound that activates PARP, induces necrosis,and/or is an anti-cancer agent). As discussed, herein, the PARPactivation and/or necrosis can be measured in the absence and thepresence of the test compound. In some embodiments, the positive controlis MNNG.

The present invention also provides methods of treating cancer in anindividual. In some embodiments, the methods comprise identifying ananti-cancer agent according to the methods described herein andadministering the agent to the individual. In some embodiments, theanti-cancer agent is co-administered with at least one other cancertreatment to the individual. In some embodiments, the other cancertreatment is a tumor apoptosis inducing agent. In some embodiments,apoptosis inducing agent is a bcl-2 inhibitor.

The anti-cancer agent can be administered by any means to theindividual. Methods of administration are known to one of skill in theart. For example, the anti-cancer agent can be prepared as apharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier or diluent.In some embodiments, the pharmaceutical compositions are sterile and/orpyrogen free. The pharmaceutical composition comprising the anti-canceragent and a pharmaceutically acceptable carrier or diluent may beformulated by one having ordinary skill in the art with compositionsselected depending upon the chosen mode of administration. Suitablepharmaceutical carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, A. Osol, a standard reference textin this field.

For parenteral administration, the anti-cancer agent can be, forexample, formulated as a solution, suspension, emulsion or lyophilizedpowder in association with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 5% human serum albumin. Liposomes and nonaqueousvehicles such as fixed oils may also be used. The vehicle or lyophilizedpowder may contain additives that maintain isotonicity (e.g., sodiumchloride, mannitol) and chemical stability (e.g., buffers andpreservatives). The formulation is sterilized by commonly usedtechniques. For example, a parenteral composition suitable foradministration by injection is prepared by dissolving 1.5% by weight ofactive ingredient in 0.9% sodium chloride solution.

The pharmaceutical compositions according to the present invention maybe administered as a single doses or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The pharmaceutical compositions comprising anti-cancer agent may beadministered by any means that enables the agent to reach the agent'ssite of action in the body of a mammal. Because anti-cancer agents maybe subject to being digested when administered orally, parenteraladministration, i.e., intravenous, subcutaneous, intramuscular, wouldordinarily be used to optimize absorption. In addition, thepharmaceutical compositions of the present invention may be injected ata site at or near hyperproliferative growth. For example, administrationmay be by direct injection into a solid tumor mass or in the tissuedirectly adjacent thereto. The composition may also be formulated with apharmaceutically acceptable topical carrier and the formulation may beadministered topically as a creme, lotion or ointment for example.

The dosage administered varies depending upon factors such as:pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment. Usually, adaily dosage of the anti-cancer agent is an amount effect to activatePARP sufficiently to have an anti-cancer effect. In some embodiments,the dosage can be about 1 μg to 100 milligrams per kilogram of bodyweight. Ordinarily 0.5 to 50, and preferably 1 to 10 milligrams perkilogram per day given in divided doses 1 to 6 times a day or insustained release form is effective to obtain desired results.

In some embodiment, the present invention relates to kits for practicingthe above described method of identifying and/or detecting compoundsthat activate PARP, induce necrosis, or can act as anti-cancer agents.Kits according to this aspect of the invention comprises the a firstcontainer comprising cells that are to be contacted with test compound,optionally a second container comprising a positive control, andoptionally a third container comprising a negative control .Additionally, to practice the above defined method, means are requiredto measure PARP activation, necrosis, and/or anti-cancer activity. Insome embodiments of this aspect of the invention, a fourth containercomprising an antibody that detects PARP activation is provided. Atleast one of the contained components, for example, the antibody, may beconjugated with an agent, which allows its presence to be detected. Inthe kits of the invention which are useful to practice the methods ofidentifying compounds that activate PARP, induce necrosis, and/or haveanti-cancer activity cells that are deficient in the bax gene, bak gene,or both are provided.

In some embodiments, the kit comprises an instruction manual thatdirects the user of this kit how to use the kit. In some embodiments,the instruction manual comprises illustrations, diagrams, charts,graphs, or photographs of exemplary results from when a positive and/ornegative control is tested for PARP activation. In some embodiments, thekit comprises means for inhibiting the expression or activity of bakgene expression, bax gene expression, or both. In some embodiments, thekit comprises means for inhibiting apoptosis. In some embodiments, thekit comprises at least one oligonucleotide that can be used to inhibitthe expression of a gene that is essential for apoptosis, necrosis, orboth.

EXAMPLES Example 1 Alkylating Agents Induce Cell Death Independent ofApoptotic Effectors

Mouse embryo fibroblasts (MEFs) generated from wild-type, p₅₃ ^(−/−),and bax^(−/−)bak^(−/−) animals were tested for sensitivity to thealkylating agents mechlorethamine hydrochloride (nitrogen mustard) andN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), as well as to the kinaseinhibitor staurosporine and the DNA topoisomerase inhibitor etoposide.Staurosporine induced cell death in wild-type and p53^(−/−) cells, butnot in bax^(−/−)bak^(−/−) cells (FIG. 1, Panel A). Bothbax^(−/−)bak^(−/−) and p53^(−/−) cells were resistant to etoposidetreatment (FIG. 1, Panel B). In contrast, both bax^(−/−)bak^(−/−) andp53^(−/−) cells were as sensitive as wild-type cells to nitrogen mustardand MNNG (FIG. 1, Panels C and D). These findings indicate that DNAalkylation initiates cell death in a manner that is independent of theapoptotic initiator p53 or Bax/Bak. Additional experiments confirmedthat neither overexpression of the anti-apoptotic protein Bcl-x_(L) noraddition of the caspase inhibitors zVAD and BAF could inhibit cell deathinduced by MNNG (data not shown).

Example 2 Activation of PARP and Depletion of NAD and ATP in Response toDNA Damage

DNA alkylation has been shown to activate the enzymatic activity ofPARP, which catalyzes the synthesis of poly(ADP-ribose) polymers onhistones and other chromatin-associated proteins in the vicinity of theDNA adduct (D'Amours et al. 1999). This process promotes the efficientrecognition of the DNA damage by DNA repair enzymes. Becauseβ-nicotinamide adenine dinucleotide (NAD) is the substrate forpoly(ADP-ribosyl)ation, PARP activation has been shown to depletecellular NAD and contribute to cell death in response to excitotoxicstimuli or reperfusion injury (Szabo and Dawson 1998; Pieper et al.1999b). We tested whether PARP activation is involved in death ofbax^(−/−)bak^(−/−) cells in response to DNA damage. Upon MNNG treatment,PARP was activated equally in wild-type and bax^(−/−)bak^(−/−) cells, asindicated by the increase of poly(ADP-ribose) polymers (PAR) (FIG. 2,Panel A). MNNG treatment also caused a dose-dependent decrease in NADand ATP in both wild-type and bax^(−/−)bak^(−/−) cells. This depletioncould be prevented by nicotinic acid analogue DPQ which among otherfunctions acts in vitro as a PARP inhibitor (FIG. 2, Panels B and C).

Example 3 DNA Damage-induced Necrosis is PARP-dependent

The above findings suggest that DNA alkylating agents can trigger thePARP-dependent depletion of NAD and ATP in cells, and this process isindependent of the mitochondrial apoptosis pathway. To test directly ifPARP is required to initiate NAD/ATP depletion and cell death inresponse to MNNG, shRNA was used to suppress the expression of PARP-1,which accounts for ˜90% of the PARP activity among the PARP familymembers (Smith 2001). Among the stable bax^(−/−)bak^(−/−) MEF clonestransfected with an shRNA vector, Clone HP17 demonstrated a dramaticreduction of PARP-1 expression, while in Clone HP11 the PARP-1 level wasmoderately decreased (FIG. 3, Panel A). Correlating with the levels ofPARP-1 expression, poly(ADP-ribosyl)ation was moderately decreased inclone HP11 upon MNNG treatment, and significantly decreased in cloneHP17 (FIG. 3, Panel B). The remaining poly(ADP-ribosyl)ation may resultfrom minimal residual PARP-1 which could not be detected byimmunoblotting, or from the activation of other PARP family members(Smith 2001). Despite the residual PARP activity in Clone HP17, ATPlevels were maintained upon MNNG treatment (FIG. 3, Panel C).Importantly, no cell death was observed in clone HP17 when treated withMNNG at concentrations that killed all vector control bax^(−/−)bak^(−/−)cells (FIG. 3, Panel D).

In contrast to bax^(−/−bak) ^(−/−) cells, the death of wild-type cellswas only partially rescued by PARP inhibitor DHIQ, suggesting that DNAalkylators can induce more than one death pathway. To test this further,wild-type, bax^(−/−)bak^(−/−), parp-1^(−/−) (Wang et al. 1995), andClone HP17 (bax^(−/−)bak^(−/−), shPARP-1) cells were compared side byside for sensitivity to different death stimuli (FIG. 3, Panels E andF). All wild-type and bax^(−/−)bak^(−/−) cells died within 20 hoursfollowing 0.5 mM MNNG treatment. In contrast, only 30% of similarlytreated parp-1^(−/−) cells died and HP17 cells showed essentially nocell death (FIG. 3, Panels E and F). The reduced cell density observedresulted from the growth arrest effect caused by DNA alkylation.Wild-type and parp-1^(−/−) cells were killed by staurosporine, while nocell death was observed in bax^(−/−)bak^(−/−) and clone HP17 cells (FIG.3, Panel F). The PARP inhibitor DHIQ blocked the cell death partially inwild-type cells, and fully in bax^(−/−)bak^(−/−) cells. DHIQ did notaffect the cell death rate in parp-1^(−/−) cells (FIG. 3, Panel F).While additional MNNG-treated parp-1^(−/−) cells underwent apoptosisover the next several days in culture, over 30% of cells treated with0.25 mM MNNG were alive three days after the treatment, and almost 90%of HP17 cells remained viable (FIG. 3, Panel G). Taken together, thesefindings indicate that two independently regulated death pathways can betriggered in response to alkylating DNA damage. One is mediated via theBax/Bak mitochondrial gateway accounting for the cell death observed inparp-1^(−/−) cells and the DHIQ-insensitive death in the other cells.The other form of death is mediated by PARP activation and isBax/Bak-independent.

Example 4 Cell Death in the Absence of Bax and Bak is Necrotic

To test if PARP-dependent cell death in response to DNA alkylatingagents was distinguishable from apoptosis, morphologic features of thecell death in response to MNNG and staurosporine were characterized byelectron microscopy. Both wild-type and bax^(−/−)bak^(−/−) cellsacquired morphologic changes characteristic of necrosis upon MNNGtreatment. These included organelle swelling, intracellular vacuoleformation, plasma membrane disintegration, and nuclear degradationwithout condensation (FIG. 4, Panel A). In addition, some wild-typecells displayed an intermediate morphology with both apoptotic andnecrotic features. In contrast, staurosporine induced apoptoticmorphological changes in wild-type cells including condensed chromatinand no obvious disintegration of the cell body. Although staurosporinetreatment induced some non-specific changes in the appearance ofbax^(−/−)bak^(−/−) cells, apoptotic features were not observed and thecells remained viable (FIG. 4, Panel A).

A central event in apoptosis is the release of apoptogenic factors suchas cytochrome c from the mitochondrial intermembrane space into thecytosol. In bax^(−/−)bak^(−/−) cells treated with MNNG, no change ofcytochrome c distribution pattern was observed, although the cells hadundergone cell death as indicated by shrinkage of nuclei (FIG. 4, PanelB). In addition, bax^(−/−)bak^(−/−) cells displayed none of thebiochemical apoptotic hallmarks tested, including caspase cleavage ofPARP-1 and lamin B1 (FIG. 4, Panel C). It is interesting to note that inwild-type cells, treatment with MNNG resulted in some cells acquiringapoptotic features. Cytochrome c release was observed in some cells(FIG. 4, Panel B), and a decrease in the caspase substrates cleavage ofPARP-1 and lamin B1 observed in the population as a whole (FIG. 4, PanelC). Furthermore the caspase cleaved forms of PARP-1 and lamin B1 wereobserved in the wild-type population primarily in the presence of PARPinhibitors (FIG. 4, Panel C). This indicated that MNNG may trigger bothnecrotic and apoptotic responses in wild-type cells. The apoptoticcomponent of the death is not dependent on PARP, since the apoptoticfeatures persisted when PARP inhibitors DHIQ and DPQ were applied (FIG.4, Panels B and C).

Example 5 PARP-Mediated Cell Death is Proinflammatory

Apoptotic cells die in an ordered fashion, and are engulfed and clearedin vivo, whereas necrotic cells lose their membrane integrity andrelease the cellular contents into the extracellular environmenttriggering an acute inflammatory response. One of the proinflammatorymolecules reported to be released into the extracellular environmentduring necrotic cell death is HMGB1, a chromatin-associated protein thatif released from cells acts as a ligand for the monocyte/macrophagescavenger receptor RAGE (Scaffidi et al. 2002). MNNG-treated cells wereevaluated for HMGB1 localization. In untreated cells, HMGB1 localized tothe nucleus. However, 6 hours following treatment of MNNG there wastranslocation of HMGB1 from nucleus to the cytosol (FIG. 5, Panel A).This redistribution was active, as it began prior to observable celldeath. By 16 hours after MNNG treatment, HMGB1 could be found in theextracellular environment. HMGB1 redistribution and release was blockedwhen PARP was inhibited (FIG. 5, Panel B). To determine if the releaseof factors such as HMGB1 is sufficient to induce an inflammatoryresponse in innate immune cells, cell culture medium was collected andadded to cultured macrophages. Macrophage activation was assessed by theproduction of the proinflammatory cytokine TNFα. Culture medium fromMNNG treated wild-type and bax^(−/−)bak^(−/−) cells induced TNFαproduction, whereas medium harvested from apoptotic cells (wild-type andparp-1^(−/−) treated with staurosporine) failed to do so (FIG. 5, PanelC). Taken together, these findings indicate that MNNG induces necroticcell death, and that the dying cell actively establishes its ability torelease inflammatory mediators upon death.

Example 6 Proliferating Cells are More Sensitive to PARP-mediatedNecrosis

In vivo, chemotherapeutic agents selectively induce the death of tumorcells and normal cells undergoing cell division (DeVita 1997). We nextinvestigated whether MNNG-induced cell death might account for suchselectivity. Since both apoptotic and necrotic death pathways can beactivated by DNA damage, we took advantage of bax^(−/−)bak^(−/−) cellsto study PARP-mediated necrosis without interference from apoptosis.Interleukin-3 (IL-3)-dependent bax^(−/−)bak^(−/−) cells were utilized.Upon IL-3 deprivation, these cells withdraw from the cell cycle butremain viable. The bax^(−/−)bak^(−/−) cells proliferating in response toIL-3 were killed by MNNG in a dose-dependent manner. In contrast,IL-3-deprived bax^(−/−)bak^(−/−) cells were resistant to MNNG (FIG. 6,Panel A). Furthermore, this protection was not transient. When IL-3 wasadded to the IL-3-dependent cultures 24 hour after 0.5 mM MNNGtreatment, cells recovered and cell viability in the culture 48 hourslater was reproducibly greater than 85%. In contrast, the viability ofcells treated with 0.5 mM MNNG in the presence of IL-3 continued todecline over the first 72 hours after treatment. Similarly, we observedthat MEFs grown to confluence and then serum-deprived were lesssensitive to MNNG-induced cell death than subconfluent MEFs cultured inthe presence of serum (FIG. 6, Panel B). This suggested that growthfactor signal transduction and/or cell cycle commitment might contributeto sensitivity to PARP-mediated necrosis. To determine whether thedecreased susceptibility of non-proliferating cells to MNNG is due toimpaired activation of PARP, PARP activation was determined. Equivalentincreases in poly(ADP-ribosyl)ation were observed in vegetative andproliferating cells (FIG. 6, Panel C). Based on assaying isolated DNAfor strand breaks, equivalent amounts of DNA damage were observed incells growing in the presence or absence of IL-3. Consistent with theincrease of poly(ADP-ribosyl)ation, cellular NAD level was decreased toa similar extent under both culture conditions (FIG. 6, Panel D).However, while the total cellular ATP level was drastically decreased incells cultured in the presence of IL-3, the ATP level was preserved inthe cells deprived of IL-3 (p<0.01, FIG. 6, Panel D).

Two major ATP generation mechanisms exist in a cell involving NADutilization: one through glycolysis using cytosolic NAD as a substrate,and another by mitochondrial oxidative phosphorylation via the F₁F₀-ATPsynthase. Thus, NAD consumption by PARP would be predicted to have adifferential effect on glycolysis and oxidative phosphorylation sincecytosolic NAD and mitochondrial NAD do not exchange with each other.Consistent with this, subcellular fractionation showed that while MNNGtreatment reduced cytosolic NAD to minimal levels, mitochondrial NAD wasnot significantly affected (FIG. 6, Panel E).

Example 7 PARP-Mediated Necrosis is Controlled by Cellular MetabolicStatus

The cytosolic and mitochondrial NAD pools are used in different pathwaysto generate ATP, namely glycolysis and oxidative phosphorylation,respectively. The finding that PARP preferentially depletes cytosolicNAD suggested that cells sensitive to PARP might depend on glucosemetabolism for ATP production. Consistent with this model, IL-3 treatedcells were found to be highly glycolytic (FIG. 7, Panel A) andinhibition of oxidative phosphorylation did not affect the ATP level ofthese cells (FIG. 7, Panel B). In contrast, IL3-deprived cells displayeda low glycolytic rate (FIG. 7, Panel A, Gonin-Giraud et al. 2002) andATP levels fell dramatically when oxidative phosphorylation wasinhibited with oligomycin (FIG. 7, Panel B). Together, these datasuggest that growth factor status affects glycolytic rate and thepredominant means of ATP production.

In IL-3-stimulated cells ATP falls when cytosolic NAD is depleted (FIG.6, Panel D). This suggests that these cells are unable to increaseoxidative phosphorylation to maintain ATP when NAD depletion compromisesthe ability to carry out glycolysis. A potential explanation for this isthat in response to IL-3-induced growth, cells shunt amino acids andlipids away from ATP-generating oxidative metabolism and into syntheticpathways. Under conditions of declining extracellular glucoseavailability, IL-3-stimulated cells can upregulate mitochondrial fattyacid oxidation to generate ATP, a process not dependent on cytosolicNAD. To determine whether this might have a protective effect, wewithdrew IL-3-treated cells from glucose over 48 hours before treatmentwith MNNG. When IL-3 stimulated cells were adapted to low glucose, theydisplayed resistance to MNNG-induced death that was comparable to thatof IL-3-deprived cells (FIG. 7, Panel C). Thus IL-3 signal transductionalone is not sufficient to confer sensitivity to MNNG-induced celldeath.

Finally, to determine if PARP-induced death occurs as a result ofglycolytic blockade, we tested whether PARP-induced necrosis could besuppressed by supplying the cells with the glycolytic end productpyruvate. baxl^(−/−)bak^(−/−) cells growing in the presence of IL-3 weresupplemented with 10 mM methyl-pyruvate, a membrane permeable form ofpyruvate, and then treated with MNNG. Methyl-pyruvate supplementationrendered IL-3-stimulated bax^(−/−)bak^(−/−) cells as resistant toMNNG-induced cell death as IL-3-deprived cells (FIG. 7, Panel C). Thus,cells utilizing substrates that are catabolized in the mitochondria tomaintain ATP production are resistant to MNNG-induced necrosis. Todemonstrate that the resistance to MNNG-induced necrosis was not apeculiar event in bax^(−/−)bak^(−/−) cells, cells with wild-type Bax andBak were pre-incubated with methyl-pyruvate before MNNG treatment.Similar to bax^(−/−)bak^(−/−) cells, methyl-pyruvate treatment of cellswith wild-type Bax and Bak also increased their resistance toMNNG-induced cell death (FIG. 7, Panel D). Thus, cellular metabolicstatus does not only determine the cell fate in Bax/Bak null background,but also contributes to the regulation of cell fate in cells capable ofundergoing Bax/Bak-dependent apoptosis in response to DNA damage.

Example 9 Materials and Methods

Cell Culture and Cell Death Assay

MEFs and BMK cells were cultured as described (Zong et al. 2001;Degenhardt et al. 2002a). IL-3-dependent bax^(−/−)bak^(−/−) cells werecultured in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 1%penicillin/streptomycin, 20 mM HEPES, 50 μLM 2-mercaptoethanol,supplemented with 4 ng/ml recombinant mouse IL-3 (BD-Pharmingen). IL-3dependent FL5.12 cells were cultured in the same media as used forbax^(−/−)bak^(−/−) cells, supplemented with 0.4 ng/ml recombinant mouseIL-3. To induce cell death with MNNG (Sigma), cells were treated withMNNG for 15 min. Cells were washed and fed with fresh medium with noMNNG, and cultured for indicated periods of time. For etoposide(Calbiochem), staurosporine (Sigma), and mechlorethanmine hydrochloride(nitrogen mustard, Sigma) treatment, the agents remained in the medium.PARP inhibitors DPQ or DHIQ (Sigma) were kept in the medium throughoutthe course of experiment when used. At the end of experiments, propidiumiodide (PI, 1 μg/ml, Molecular Probes) was added to the cells. Celldeath was determined using flow cytometry by PI exclusion.

Immunoblotting and Immunofluorescence

Cells were lysed in RIPA buffer (1% Sodium Deoxycholine, 0.1% SDS, 1%Triton X-100, 10 mM Tris pH8.0, 0.14 M NaCl) with protease inhibitorcomplex (Roche). 20 μg of protein was loaded on pre-cast 4-12% NuPAGEgel. Western blotting was performed with the following antibodies: PARP(clone C2-10, BD-Pharmingen or Trevigen), Lamin B1 (M-20, Santa Cruz),Tom20 (FL-145, Santa Cruz), PAR (BD-Pharmingen), COX IV (MolecularProbes), Tubulin (Sigma), and HMGB1 (BD-Pharmingen). Forimmunofluorescence, cells were fixed in 4% paraformaldehyde andpermeabilized for 10 min in PBS containing 0.2% Triton X-100. Cells werewashed with PBS containing 0.02% Triton X-100 and 1.5% FBS, followed byincubation with antibodies against Bax (6A7, BD-Pharmingen), cytochromec (BD-Pharmingen), or HMGB1 for 1 h at room temperature. Cells wereincubated with Rhodamine-conjugated secondary antibody (JacksonImmunoResearch). Nuclei were visualized by staining with 1 μg/ml DAPI.Images were captured on a 510 LSM confocal microscope (Zeiss).

Determination of NAD and ATP

The concentration of NAD was measured as described (Jacobson andJacobson 1976) with modification. Briefly, 1×10⁵ cells were trypsinizedand resuspended in 100 μl 0.5 M perchloric acid. Cell extracts wereneutralized with equal volume of 1 M KOH and 0.33 M KH₂PO₄/K₂HPO₄ pH7.5. After centrifugation to remove the KClO₄ precipitate, 200 μl of NADreaction mixture (600 mM ethanol, 0.5 mM 3-[4,5dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT), 2 mMphenazine ethosulfate, 5 mM EDTA, 1 mg/ml BSA, 120 mM bicine, pH 7.8)was added to 50 μl of the supernatant or NAD standard and incubated at37° C. for 5 min. 25 μl of alcohol dehydrogenase (0.5 mg/ml in 100 mMbicine, pH7.8) was added to the reaction and incubated at 37° C. for 20min. 250 μl of 12 mM iodoacetate was added to stop the reaction, and ODwas read at 570 nm wavelength. Protein concentration was determinedusing BCA protein assay reagents (Pierce). The content of NAD wasnormalized by protein content. For ATP determination, 2×10⁴ cells werelysed in a cell lysis reagent (Catalog #1699709, Boehringer Mannheim).ATP concentration was determined as previously described (Vander Heidenet al. 1999).

Transmission Electronic Microscopy

Cells were rinsed with serum-free DMEM medium, and fixed with pre-warmed2.5% glutaraldehyde, 2% formaldehyde in 0.1 M sodium cacodylate bufferfor 1 h. Cells were washed, post-fixed with 2% osmium tetroxide,dehydrated with ascending grades of ethanol and propylene oxide, andembedded in LX-112 medium (Ladd, Vt.). After polymerization, ultrathin(90 nm) sections were cut with a diamond knife, collected on uncoatedcopper grids, and stained with uranyl acetate (1%) and lead citrate(0.2%). Samples were examined with a JEOL-1010 electron microscope(JEOL, Japan) operated at 80 KV.

shRNA

A forward oligo with the sequence from a RNA Polymerase III specific U6promoter CAG TGG AAA GAC GCG CAG GCA (SEQ ID NO:1), and a reverse oligoAAA AAA GGA AGT GAA AGC GGC CAA CGT TCC TCG AGC AAC GTT GGC CGC TTT CACTTC CG TGT TTC GTC CTT TCC ACA A (SEQ ID NO: 2) that contains a hairpinof the PARP-1 transcript sequence and sequence from the U6 promoter,were used in a PCR to generate a DNA fragment that allows thetranscription of the PARP-1 hairpin under the control of the U6promoter. This DNA cassette was cloned into a pBabe-puro basedretroviral vector. The control vector or PARP-1 hairpin constructs weretransfected into MEFs using Lipofectamine 2000 transfection reagent(Invitrogen). Single cell clones were propagated using limited dilution,and screened for their PARP-1 expression.

TNFα Measurement

Mouse bone marrow derived macrophages were generated as described (Myunget al. 2000). 5×10⁵ MEFs were plated in 6-well plates, and treated withMNNG for 15 min or staurosporine for 2 h. Cells were washed and refedwith 1 ml fresh culture media. 16 h later the cell culture media werecollected and added to 1×10⁵ macrophages/well plated in 96-well plates aday earlier. TNFα production was measured 24 h later using a QuantikineTNFα ELISA kit (R&D Systems).

Subcellular Fractionation

IL-3-dependent cells were suspended in hypotonic Buffer A (250 mMsucrose, 20 mM Hepes (pH7.5), 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mMEGTA, 1× protease inhibitor complex (Roche)). Subcellular fractionationwas carried out as previously described (Zong et al. 2003). Theresulting cytosolic fraction was used directly for NAD measurement,protein quantification, and immunoblotting. The mitochondrial pellet wasdivided into two equal parts, one was lysed in 0.5 mM HClO₄, and usedfor NAD measurement, and another part was lysed in RIPA buffer forprotein quantification and immunoblotting.

Measurement of Glycolysis

Cellular glycolysis rate was determined as described (Liang et al. 1997)with modifications. One million cells were incubated with 10 μCi5-³H-glucose (PerkinElmer Life Sciences) at 37° C. for 1 h. Followingincubation, the reaction was stopped with 0.2 N HCl and ³H₂O wasseparated from 5-³H-glucose by diffusion in an airtight container.Diffused and undiffused tritium was measured using a 1450 Microbetascintillation counter (Wallac) and compared to controls of 5-³H-glucoseonly and ³H₂O only to determine the rate of glycolysis.

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Those skilled in the art will appreciate that numerous changes andmodifications may be made to the embodiments of the invention and thatsuch changes and modifications may be made without departing from thespirit of the invention. It is therefore intended that the appendedclaims cover all such equivalent variations as fall within the truespirit and scope of the invention. Also, it is intended that each of thepatents, patent applications, and publications referenced above beincorporated by reference herein.

1. A method of identifying a compound which can induce cell death bynecrosis in cells which rely on catabolizing glucose for ATP productionin vitro comprising the steps of: 1) contacting a plurality ofimmortalized cells in vitro that are deficient in Bax gene expressionand Bak gene expression with a test compound; 2) determining if PARP isactivated in said cells contacted with said test compound wherein theactivation of PARP in said cells contacted with said test compoundindicates that the test compound can induce cell death by necrosis incells in vitro which rely on catabolizing glucose for ATP production. 2.The method of claim 1, wherein said measuring PARP activation comprisesmeasuring PAR polymers, NAD depletion, or ATP depletion.
 3. The methodof claim 1, further comprising performing a negative control assay whichcomprises determining if PARP is activated in a plurality ofbax^(−/−)bak^(−/−) immortalized cells in the absence of the testcompound.
 4. The method of claim 1, further comprising performing apositive control assay which comprises determining if PARP is activatedin a plurality of bax^(−/−)bak^(−/−) immortalized cells with a positivecontrol compound and determining if PARP is activated.
 5. The method ofclaim 1, wherein the PARP activation in said cells contacted with saidtest compound is at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, or at least 200% greater than the PARP activation in saidcells in the absence of said test compound.
 6. A method of identifying acompound which can induce cell death by necrosis in cells which rely oncatabolizing glucose for ATP production in vitro comprising, the stepsof: 1) contacting a plurality of immortalized cells in vitro that aredeficient in Bax gene expression and Bak gene expression with a testcompound; 2) determining if PARP is activated in said cells contactedwith said test compound; 3) determining if necrosis is induced in saidcells; wherein the activation of PARP and induction of necrosis in saidcells contacted with said test compound indicates that the testcompound-can induce cell death by necrosis in cells in vitro which relyon catabolizing glucose for ATP production.
 7. The method of claim 6,wherein said measuring PARP activation comprises measuring PAR polymers,NAD depletion, or ATP depletion.
 8. The method of claim 6, furthercomprising performing a negative control assay which comprisesdetermining if PARP is activated in a plurality of bax^(−/−)bak^(−/−)immortalized cells in the absence of the test compound.
 9. The method ofclaim 6, further comprising performing a positive control assay whichcomprises determining if PARP is activated in a plurality ofbax^(−/−)bak^(−/−) immortalized cells with a positive control compoundand determining if PARP is activated.
 10. The method of claim 6, whereinthe PARP activation in said cells contacted with said test compound isat least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, orat least 200% greater than the PARP activation in said cells in theabsence of said test compound.
 11. The method of claim 6, whereinnecrosis is determined by measuring organelle swelling, intracellularvacuole formation, plasma membrane disintegration, or nucleardegradation without condensation.
 12. A method of identifying a compoundthat can induce necrosis in cells which rely on catabolizing glucose forATP production in vitro comprising: 1) contacting a plurality ofimmortalized cells in vitro that are deficient in Bax gene expressionand Bak gene expression with a test compound; and 2) determining ifnecrosis is induced in said cells contacted with said test compound;wherein induction of necrosis in said cells contacted with said testcompound indicates that the test compound-can induce cell death bynecrosis in cells in vitro which rely on catabolizing glucose for ATPproduction.
 13. The method of claim 12, wherein necrosis is determinedby measuring organelle swelling, intracellular vacuole formation, plasmamembrane disintegration, or nuclear degradation without condensation.